PMN recruitment and sequestration to sites of inflammation and second organ injury is initiated by pro-inflammatory mediators, among which LTB4 is considered to be very important (Weissmann, G., Smolen, J. E., and Korchak, H. M. 1980. Release of inflammatory mediators from stimulated neutrophils. N. Engl. J. Med. 303: 27-34; Sammuelsson, B. 1983. Leukotrienes: Mediators of inflammation and immediate hypersensitivity. Science 220: 568-575). Recently, the LTB4 receptor (BLTR) was also shown to serve as a coreceptor for HIV-1 entry, which further emphasizes the crucial role of this system in host defense (Owman, C. 1998. The leukotriene B4 receptor functions as a novel type of coreceptor mediating entry of primary HIV-1 isolates into CD4-positive cells. Proc. Natl. Acad. Sci. USA 95: 9530-9534). Aspirin is widely used for its anti-inflammatory and analgesic properties with several newly identified therapeutic actions including prevention of cardiovascular diseases and decreasing incidence of lung, colon and breast cancers, which increases the importance of obtaining complete knowledge of aspirin""s mechanism of action (Marcus, A. J. 1995. Aspirin as prophylaxis against colorectal cancer. N. Eng. J. Med. 333: 656-658). When aspirin is given, in addition to inhibiting prostanoid biosynthesis, it also triggers the endogenous transcellular production of 15 epimeric or 15R LXA4, termed aspirin-triggered LXA4 (ATL), which appears to mediate in part some of aspirin""s therapeutic impact and is generated in vivo (Serhan, C. N. 1997. Lipoxins and novel aspirin-triggered 15-epi-lipoxins (ATL). Prostaglandins 53: 107-137; Chiang, N., Takano, T., Clish, C. B., Petasis, N. A. and Serhan C. N. 1998. Aspirin-triggered 15-epi-Lipoxin A4 (ATL) generation by human leukocytes and murine peritonitis exudates: development of a specific 15-epi-LXA4 ELISA. J. Phar. Exp. Ther. 287: 779-790). LXA4 controls leukocyte responses via its own specific G protein coupled receptor, denoted ALXR, which also engages 15-epi-LXA4 (Serhan, C. N. 1997. Lipoxins and novel aspirin-triggered 15-epi-lipoxins (ATL). Prostaglandins 53: 107-137; Takano, T., Clish, C. B., Gronert, K., Petasis, N. A. and Serhan C. N. 1997. Neutrophil-mediated changes in vascular permeability are inhibited by topical application of aspirin-triggered 15-epi-lipoxin A4 and novel lipoxin B4 stable analogues. J. Clin. Invest. 101: 819-826). Like other local mediators, LXs are rapidly generated, evoke responses and are inactivated by further metabolism (Serhan, C. N. 1997. Lipoxins and novel aspirin-triggered 15-epilipoxins (ATL). Prostaglandins 53: 107-137). Methods to monitor, study and screen potential pharmaceuticals, e.g., antiinflammatories, which interact with the identified receptors, therefore, are of interest.
In one aspect the present invention pertains to a non-human transgenic mammal that produces in its leukocytes a recombinant human leukotriene B4 receptor (BLTR) having physiological activity of human BLTR. The transgenic mammal has stably integrated into its genome an exogenous gene construct which includes (A) 5xe2x80x2 expression regulating sequences, including a BLTR specific promoter, (B) DNA encoding the BLTR and a signal sequence effective in directing overexpression of the BLTR into leukocytes of the transgenic mammal and (C) 3xe2x80x2 regulatory sequences that result in the overexpression of the DNA in the leukocytes. In one embodiment, (A), (B), and (C) are operably linked in the gene construct to obtain production of the BLTR in the leukocytes and overexpression thereof in the transgenic mammal.
In another aspect, the invention pertains to a process for producing recombinant human BLTR having physiological activity of human BLTR. The method includes providing a non-human transgenic mammal having integrated into its genome an exogenous gene construct that includes (A) 5xe2x80x2 expression regulating sequences, including a BLTR specific promoter, (B) DNA encoding the BLTR and a signal sequence effective in directing overexpression of the BLTR in leukocytes of the transgenic mammal and (C) 3xe2x80x2 regulatory sequences that result in the overexpression of the DNA in the leukocytes. In one embodiment, (A), (B), and (C) are operably linked in the gene construct to obtain production of the BLTR in the leukocytes and overexpression thereof in the transgenic mammal. The BLTR is overexpressed and secreted in the leukocytes of the transgenic mammal.
In yet another aspect, the present invention pertains to a process for producing a non-human transgenic mammal that produces in its leukocytes a recombinant human leukotriene B4 receptor (BLTR) having physiological activity of human BLTR. The method includes (a) providing an exogenous gene construct that includes (A) 5xe2x80x2 expression regulating sequences, including a BLTR specific promoter, (B) DNA encoding the BLTR and a signal sequence effective in directing overexpression of the BLTR in leukocytes of the transgenic mammal and (C) 3xe2x80x2 regulatory sequences that result in the overexpression of the DNA in the leukocytes. In one embodiment, (A), (B), and (C) are operably linked in the gene construct to obtain production of the BLTR in the leukocytes and overexpression thereof in the transgenic mammal. The construct of step (a) is introduced into a non-human mammalian embryo, wherein the construct is stably integrated into the genome of the mammalian embryo. The embryo is allowed to develop into a non-human transgenic mammal and it is determined whether the non-human transgenic mammal of (c) overexpresses BLTR.
In still another aspect, the present invention pertains to a method for screening compounds that inhibit overexpression of polymorphonuclear leukocyte production in a non-human transgenic mammal that produces in its leukocytes a recombinant human leukotriene B4 receptor (BLTR) having physiological activity of human BLTR. The method includes (a) providing a non-human transgenic mammal having integrated into its genome an exogenous gene construct which includes (A) 5xe2x80x2 expression regulating sequences, including a BLTR specific promoter, (B) DNA encoding the BLTR and a signal sequence effective in directing overexpression of the BLTR into leukocytes of the transgenic mammal and (C) 3xe2x80x2 regulatory sequences that result in the overexpression of the DNA in the leukocytes. In one embodiment, (A), (B), and (C) are operably linked in the gene construct to obtain production of the BLTR in the leukocytes and overexpression thereof in the transgenic mammal. The BLTR is allowed to be overexpressed and secreted in the leukocytes of the transgenic mammal. The transgenic mammal can be subjected to physiological stressing, thereby causing increased neutrophil recruitment to the leukocytes via the overexpressed BLTR. A therapeutically effective amount of a compound is administered to the mammal to interact with the neutrophil recruitment in response to the stress. It is then determined empirically, whether the compound, e.g., an lipoxin derivative, reduces neutrophil activation in the leukocytes of the transgenic mammal which have overexpress BLTR therein. Alternatively, step (d) is performed prior to step (c), thereby preventing or inhibiting neutrophil activation.
In one preferred embodiment the transgenic mammal produces human BLTR. In another preferred embodiment, the human BLTR has the amino acid sequence of human BLTR. In still another preferred embodiment, the mammal is selected from the group consisting of rat, rabbit, pig, sheep, goat or cow, and most preferably a mouse. It is preferred that the mammal is a female, e.g., a female mouse.